IGF-I for myocardial repair

ABSTRACT

Provided herein are methods for treating an individual having (suffering from) an acute myocardial infarction and drug eluting stents useful for treating such individuals. These methods include treating an individual by introducing, such as by surgically inserting, at a site of an acute coronary artery occlusion upstream of the site of acute myocardial infarction, a drug eluting stent (DES) that is capable of eluting from 25 pg to 950 pg of IGF-1 directly into the coronary circulation. The treatment is specifically directed to stimulation of repair or survival of damaged cardiac muscle or left ventricular remodeling.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of currently pending U.S.Ser. No. 13/132,087, filed Jun. 13, 2011, which is a 371 National PhaseEntry Application of International Application No. PCT/IE2009/000084filed Nov. 27, 2009, which designates the U.S., and which claims benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/118,829filed Dec. 1, 2008, the contents of which are incorporated herein byreference in their entirety.

SEQUENCE LISTING

The sequence listing of the present application has been submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “Sequence-Listing-048262_070070_US”, creation date of Apr. 26,2017 and a size of 3,388 bytes. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

TECHNICAL FIELD

The invention relates to a stent suitable for implantation in themyocardial circulatory system and capable of delivering IGF-1 into themyocardial circulation. The invention also relates to a method oftreating damaged cardiac muscle, especially cardiac muscle damaged bymyocardial infarction, to stimulate survival and repair of damagedcardiac muscle, or stimulate left ventricular remodeling.

BACKGROUND TO THE INVENTION

Myocardial infarction (formation of an infarct or an area of dead heartmuscle) occurs when the blood supply to the heart is interrupted, whichcan be the result of occlusion (blockage) of a coronary artery, such asfollows the rupture of vulnerable atherosclerotic plaque. Acutemyocardial infarction (AMI) occurs as the result of sudden blockage ofblood supply to the heart. Irreversible death of heart of heart musclebegins to occur if the blood supply is not re-established quickly enough(e.g., within 20 to 40 minutes).

If impaired blood flow to the heart lasts long enough, heart cells die,via necrotic and/or apoptotic cellular pathways, do not grow back and acollagen scar forms in their place. This can result in permanent damageto the heart and scar tissue also puts the patient at risk forpotentially life threatening arrhythmias, and/or may result in theformation of a ventricular aneurysm.

Diseases of the heart, such as MI, are the leading cause of death forboth men and women. Coronary heart disease is responsible for 1 in 5deaths in the U.S. About 1.2 million people in the U.S. suffer anew orrecurrent coronary attack every year and of them, approximately 400,000of them die as a result of the attack.

STATEMENTS OF INVENTION

In one embodiment, the invention relates to a drug eluting stentsuitable for intracoronary implantation, the stent comprising a stentbody, and a coating covering at least a part of the stent body, whereinthe coating comprises IGF-1 and is capable of releasing a therapeuticdose of from 25 pg to 950 pg IGF-1 into the coronary circulation over anelution period of up to 14 days.

The invention also relates to a stent having an IGF-1 eluting coating,wherein the coating is adapted to release a therapeutic dose of from 25to 950 pg IGF-1 into the coronary circulation over an elution period ofup to 14 days.

The invention further relates to a stent, typically suitable forintracoronary implantation, and having an IGF-1 eluting coating, thecoating being loaded with a therapeutic dose of less than 1000 pg ofIGF-1, wherein the coating is typically capable of eluting at least 50%of the IGF-1 loading over an elution period of suitably up to 14 days.

Suitably, the therapeutic dose of IGF-1 is from 250 pg to 750 pg,preferably 300 pg to 700 pg, preferably 350 pg to 650 pg, morepreferably from 400 to 600 pg, more preferably from 400 to 550 pg, morepreferably from 400 to 500 pg, more preferably from 420 to 480 pg.Typically, the therapeutic dose is at least 300 pg, 350 pg, 380 pg, 390pg, 400 pg, 410 pg, 420 pg, 430 pg, or 440 pg IGF-1. Generally, thetherapeutic dose is at most 700 pg, 650 pg, 600 pg, 550 pg, 500 pg, 490pg, 480 pg, 470 pg, or 460 pg IGF-1.

In one embodiment, the coating is adapted for delivery of thetherapeutic dose of IGF-1 into the coronary circulation over a period(the elution period) of between 1 hour and 14 days, preferably between 6hours and 14 days, preferably between 12 hours and 14 days, preferablybetween 18 hours and 14 days, and preferably between 14 hours and 14days. Generally, the coating is adapted for delivery of the therapeuticdose of IGF-1 over a period of from 12 hours to 14 days, preferably from12 hours to 10 days, preferably from 12 hours to 9 days, preferably from12 hours to 8 days, and preferably from 12 hours to 7 days. Generally,the coating is adapted for delivery of the therapeutic dose of IGF-1over a period of from 20 hours to 9 days, preferably from 22 hours to 8days, preferably from 24 hours to 7 days, preferably from 36 hours to 6days, and preferably from 48 hours to 5 days.

In a preferred embodiment of the invention, the coating is adapted todeliver a therapeutic so dose of from 400 to 500 pg of IGF-1 into themyocardial circulation over a period of 12 hours to 7 days.

The term “elution period” preferably means the period for eluting thetherapeutic dose of IGF-1 into the coronary circulation. Generally, thecoating does not elute any further IGF-1 once the therapeutic dose hasbeen eluted.

In one embodiment of the invention, the coating is loaded with less than1000 pg, 900 pg, 800 pg, 700 pg, 650 pg, 600 pg, 550 pg, or 500 pg ofIGF-1. Suitably, the stent is loaded with from 300 to 900 pg, preferably350 to 850 pg, preferably 400 to 800 pg, preferably 450 to 750 pg,preferably 500 to 700 pg, of IGF-1.

In one preferred embodiment, the coating on the stent is prepared usingPEP™ technology. The details of such coating technologies, and coatingsprepared using the technology, are described in US Patent ApplicationUS2004/0241325 (Al-Lamee et al.) and generally involve priming the stentsurface with a surface functional group, and then coating thefunctionalised surface with a mixture of drug and polymer adapted toreact with the functionalised surface. The entire contents of US2004/0241325 are incorporated herein by reference. In particular, the Examples 1to 15 on pages 3 to 10 are incorporated herein by reference.

In one embodiment, the stents of the invention are adapted to deliverIGF-1 and a further cardioprotective agent into the myocardialcirculation. Examples of such further cardioprotective agents areprovided below.

In another aspect, the invention relates to a method of stimulatingsurvival or repair of cardiac muscle or left ventricular remodeling, ina mammal having damaged cardiac muscle, comprising the step ofimplanting a stent of the invention into a coronary artery of the mammalupstream of the site of the damaged cardiac muscle. Typically, themammal being treated has suffered a myocardial infarction, for examplean acute MI, although the method of treatment may be in response todamaged cardiac muscle caused by other events, for example, ischemia ortrauma.

The invention also provides a method of treating a mammal that hassuffered a myocardial infarction to stimulate repair or survival ofcardiac muscle damaged by the infarct, or to stimulate left ventricularremodeling, the method comprising a step of implanting a stent of theinvention into a coronary artery of the mammal upstream of the site ofthe damaged cardiac muscle.

The invention also relates to a method of stimulating survival or repairof cardiac muscle, or stimulating left ventricular remodeling, in amammal having damaged cardiac muscle, comprising the step ofadministering a therapeutic dose of IGF-1 to the damaged cardiac muscleby intracoronary delivery. Typically, the mammal being treated hassuffered a myocardial infarction, although the method of treatment maybe in response to damaged cardiac muscle caused by other events, forexample ischemia or trauma. Suitably, the IGF-1 is administered byintracoronary infusion into the coronary circulation, although othermethods of intracoronary delivery are envisaged such as for exampleintracoronary delivery by means of an IGF-1 eluting implantable devicesuch as a stent. Typically, a therapeutic dose of between 25 pg and 950pg IGF-1 is administered by intracoronary delivery, ideally over aperiod of from 1 second to 24 hours.

The invention also provides a method of treating a mammal that hassuffered a myocardial infarction to stimulate repair or survival ofcardiac muscle damaged by the infarct, or to stimulate left ventricularremodeling, the method comprising a step of administering a therapeuticdose of IGF-1 to the damaged cardiac muscle by intracoronary delivery.Typically, the mammal being treated has suffered a myocardialinfarction, although the method of treatment may be in response todamaged cardiac muscle caused by other events, for example ischemia ortrauma. Suitably, the IGF-1 is administered by intracoronary infusioninto the coronary circulation, although other methods of intracoronarydelivery are envisaged such as for example intracoronary delivery bymeans of an IGF-1 eluting implantable device such as a stent. Typically,a therapeutic dose of between 25 pg and 950 pg IGF-1 is administered byintracoronary delivery, ideally over a period of from 1 second to 24hours.

In the methods of the invention, the therapeutic dose of IGF-1 issuitably from 50 to 900 pg, 100 to 850 pg, 150 to 800 pg, 200 to 750 pg,300 pg to 700 pg, 350 pg to 650 pg, more preferably from 400 to 600 pg,more preferably from 400 to 550 pg, more preferably from 400 to 500 pg,more preferably from 420 to 480 pg. Typically, the therapeutic dose isat least 30 pg, 50 pg, 75 pg, 100 pg, 125 pg, 150 pg, 175 pg, 200 pg,250 pg, 300 pg, 350 pg, 380 pg, 390 pg, 400 pg, 410 pg, 420 pg, 430 pg,or 440 pg IGF-1. Generally, the therapeutic dose is at most 900 pg, 850pg, 800 pg, 750 pg, 700 pg, 650 pg, 600 pg, 550 pg, 500 pg, 490 pg, 480pg, 470 pg, or 460 pg IGF-1.

In one embodiment, the coating is adapted for delivery of thetherapeutic dose of IGF-1 into the coronary circulation over a period(the elution period) of between 1 hour and 14 days, preferably between 6hours and 14 days, preferably between 12 hours and 14 days, preferablybetween 18 hours and 14 days, and preferably between 14 hours and 14days. Generally, the coating (or stent) is adapted for delivery of thetherapeutic dose of IGF-1 over a period of from 12 hours to 14 days,preferably from 12 hours to 10 days, preferably from 12 hours to 9 days,preferably from 12 hours to 8 days, and preferably from 12 hours to 7days. Generally, the coating is adapted for delivery of the therapeuticdose of IGF-1 over a period of from 20 hours to 9 days, preferably from22 hours to 8 days, preferably from 24 hours to 7 days, preferably from36 hours to 6 days, and preferably from 48 hours to 5 days.

Suitably, the therapeutic dose is administered in a plurality of doses,for example between 1 and 100 individual doses. Ideally, the IGF-1 isdelivered by intracoronary infusion. Preferably, the IGF-1 isadministered within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours,8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 48 hours, 72 hours, ofthe cause of the damage (i.e. within 72 hours of the myocardialinfarction event). Ideally, the therapeutic dose of IGF-1 isadministered (i.e. infused) over a period of from 5 seconds to 1 hour,more preferably from 1 to 30 minutes, and even more preferably from 1 to10 minutes. The period of infusion may involve a gradual infusion of thetherapeutic dose of IGF-1, or a plurality of infusions interspersed withperiods during which the IGF-1 is not infused (to allow the infusedIGF-1 interact with the damaged cells). Thus, for example, the totalinfusion of three minutes may comprise three separate infusions of 30seconds each, separated by 30 second non-infusion periods. In oneembodiment, the IGF-1 is delivered through an angioplasty balloon.Suitably, the balloon is inflated for the periods of infusion, andtypically deflated during the non-infusion periods. It will be clearthat the therapeutic dose of IGF-1 is between and 950 pg which isrequired to be delivered to the damaged tissue over a period of time.Thus, a higher concentration of IGF-1 may be delivered over a shorterperiod of time, or a lower concentration of IGF-1 may be delivered overa longer period of time. For example, for intracoronary infusion of 450pg of IGF-1, this may be delivered by three 30 second infusions of 5 mlof IGF-1 at a concentration of 30 pg/ml.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Characterization of CPC, cytokine array of conditioned media(CM) and effect of CM on rat neonatal cardiomyocytes.

A: CPCs isolated from peripheral blood mononuclear cells werecharacterized for expression of progenitor markers using RT-PCR

B: The CM from CPC was screened for presence of about 78 differentcytokines of which the TGFβ2 and IFG1 were the predominant growthfactors.

C: Conditioned media significantly protected the rat neonatalcardiomyocytes from apoptosis induced cell death, while blocking TGFβand IGF1 using specific neutralizing antibodies abrogated the protectiveeffects of conditioned media. For neutralizing of TGFβ and IGF1, the CMwas incubated with TGFβ and/or IGF1 antibody for 15 minutes at 37° C.before exposing it to the cardiomyocytes.

D: Purified TGFβ and IGF1 peptides mimicked the effects of CM at a varynarrow dose range. The data is expressed as Mean±SEM of % Caspase 9activity to that of X-Vivo group in three independently performedexperiments.

FIG. 2. TUNEL staining in borderzone myocardium (200×).

A: Conditioned media (CM) treated group had significantly (p<0.001)reduced TUNEL signals at the borderzone myocardium compared to theX-Vivo treated group.

B: The effects of CM were blocked by neutralizing TGFβ and IGF1 in CM.The TUNEL signals from 5 sections/pig (N=3-4) with an average of 5 Highpower fields/section is expressed as Mean±SEM

C: A representative magnified (600×) image of border zone myocardiumconfirming the localization of TUNEL signal in the cardiomyocytes.

FIG. 3. Apoptotic signal in border zone myocardium 24 hrs postmyocardial infraction. Caspase 3 activity (A), Caspase 9 activity (B),and Caspase 9 protein expression (C) were significantly (p0.0001)reduced in the conditioned media treated group while the anti-apoptoticprotein BCL2 (D) was significantly upregulated. The anti-apoptoticeffects of CM are mediated by TGFβ (p0.001) and IGF1 (p<0.01). The dataare expressed as Mean±SEM of border zone myocardium samples from 3-4pigs/group.

FIG. 4. Apoptotic signal in border zone myocardium 8 weeks postmyocardial infraction. The apoptotic markers i.e., TUNEL positive nuclei(A), Caspase 9 activity (B). Caspase 3 activity (C) and BCL2 proteinexpression (D) were not effected at chronic time point (8 wks) postconditioned media therapy. The data are expressed as Mean±SEM of borderzone myocardium samples from 3-4 pigs/group.

FIG. 5. Effect of conditioned media on left ventricular infarct area.

A: Conditioned media (CM) therapy did not influence the left ventricularinfarct area at 24 hrs post MI.

B: However. CM therapy resulted in significant (P<0.01) reduction in theleft ventricular infarct area at 8 weeks post MI. The data are expressedas Mean±SEM of 6-7 ventricular cross sections/pig heart with 3-4pigs/group.

FIG. 6. Left ventricular functional analysis at 24 hrs post MI.

Conditioned media (CM) therapy significantly improved the leftventricular function i.e., ±dp/dt (A), ±dv/dt (B), stroke volume (C),and Ejection fraction (D) at 24 hrs post MI. The effects of CM appear tobe mediated by TGF and IGF1. The data are expressed as Mean±SEM 20-25cardiac cycles/pig with 3-4 pigs/group.

FIG. 7. Left ventricular functional analysis at 8 weeks post MI.

Conditioned media (CM) therapy significantly improved the leftventricular function i.e., ±dp/dt (A) ±dv/dt (B), stroke volume (C), andEjection fraction (D) at 8 weeks post MI. The data are expressed asMean±SEM 20-25 cardiac cycles/pig with 3-4 pigs/group.

FIG. 8. Effect of Conditioned media (CM) on border zone collagen contentat 24 hrs (A) and 8 weeks (B) post MI.

CM therapy significantly reduced the collagen content in the border zonemyocardium at the acute (A) but not chronic (B) time frame. The borderzone sections were stained for collagen using picrosirus red andquantified using the NIH imageJ software. Collagen staining withpicrosirus red on 5 sections/pig (n=3-4) with an average of 5 High powerfields/section is so expressed as Mean±SEM.

FIG. 9. Effects of Conditioned media (CM) on borderzone cardiomyocytehypertrophy. CM significantly (p0.001) increased border zonecardiomyocyte hypertrophy at 8 weeks post myocardial infarction and thiseffect was mediated by TGF and IGF1. High power (600×) images of borderzone cardiomyocyte were acquired using a NIKON confocal microscope andplainmetric analysis was performed using NIKON image analysis softwareto quantify cardiomyocyte size. An average of 300-400 border zonecardiomyocytes were analyzed per pig (with 3-4 pigs/group) and the dataare reported as Mean±SEM of 1000-1600 cardiomyocytes/group.

FIG. 10. Conditioned media therapy did not affect the total number ofnuclei in the border zone myocardium. Total number of DAPI positivenuclei/High power field were counted and expressed as Mean±SEM of 10sections/animal.

FIG. 11 Antiapoptotic effect of conditioned media (CM) therapy onborderzone (BZ) myocardium in vivo (TUNEL staining) was abrogated byblocking IGF-1 in CM using selective neutralizing antibody.

FIG. 12. Effect of conditioned media on left ventricular infarct area at8 weeks (A) post myocardial infarction (MI) was significantly reduced byCM therapy, which also increased thinning ratio (B) at 8 weeks post MI.

FIG. 13. Antiapoptotic effect of IGF1 (50 and 500 pg/ml) therapy onborderzone (BZ) myocardium in vivo.

DETAILED DESCRIPTION OF THE INVENTION

The application is based at least in part on the surprising finding thatcytokines, such as two cytokines that are cellular factors secreted fromendothelial progenitor cells (EPC). TGFβ1 (TGFBeta1) and IGF1, play amajor role in cardiac repair after myocardial infarction (one result ofwhich is formation of a myocardial infarct), such as acute myocardialinfarction. These factors act as cardioprotective agents in that theyexert anti-apoptotic and/or cardiotrophic and other beneficial effectson affected or diseased tissue, for example at the site of an acutemyocardial infarction. Such cytokines exert their effects, for example,by reducing or preventing apoptosis in affected or diseased tissueand/or enhancing or facilitating regional border zone hypertrophy inhealthy tissues and/or enhancing or facilitating angiogenesis in theborder zone.

Provided herein are methods of treating an individual suffering from anacute myocardial infarction, comprising administering to the individual,upstream of the myocardial infarction, a therapeutically effectiveamount of a (at least one, one or more) cardioprotective agent. Inspecific embodiments, TGFβ1, IGF1, or TGFβ1 and IGF1 are administered tothe individual, upstream of the myocardial infarction, in sufficientquantity to result in delivery to the heart of TGFβ1, IGF1 or TGFβ1 andIGF1 in concentrations that reduce effects of the myocardial infarctionon the individual (e.g., by exerting anti-apoptotic and/or cardiotrophicand other beneficial effects on affected or diseased tissue, inparticular by stimulating repair or survival of cardiac muscle orstimulating left ventricular remodeling).

In one embodiment, a therapeutically effective amount of a (at leastone, one or more) cardioprotective agent is administered to theindividual by means of a stent that comprises the cardioprotective agentand is suitably introduced at the site of an occlusion, upstream of thesite of a myocardial infarction, such as upstream of the site of acutemyocardial infarction. Typically, the stent is introduced across theocclusion. The cardioprotective agent is released from the stent insufficient quantity and at an appropriate rate to result in delivery ofthe cardioprotective agent to the heart in an amount or concentrationsufficient to assist or enhance repair of heart tissue and reduce(completely or partially) apoptosis of cardiomyocytes and, thus, reduceadverse effects on the heart. In specific embodiments in which a (atleast one, one or more) cardioprotective agent is administered by meansof a stent suitably inserted at the site of an occlusion, upstream ofthe site of acute myocardial infarction, the stent comprises (e.g., iscoated or covered with, otherwise contains) an eluting factor thatcomprises the cardioprotective agent(s). The cardioprotective agent(s)are released from the eluting factor into the myocardial circulation anddelivered to the heart via the circulation. In some embodiments, thecardioprotective agent(s) target the acute myocardial infarction borderzone, which is a region of the heart (ventricle) between/that separatesan area of grossly normal heart tissue and infarcted heart tissue. Theinfarct border zone is a site of moderately injured, partially perfused,potentially salvageable tissue, comprising myocytes, at the periphery ofdeveloping myocardial infarcts.

A stent is typically introduced, using methods known to those of skillin the art, into an individual at the time of an acute myocardialinfarction (at the time or soon after he/she has a myocardialinfarction), such as immediately, or within a few minutes to a fewhours, or within up to 72 hours after the acute infarction occurs. Forexample, the stent is introduced within 5 minutes, 10 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, or 72 hoursafter acute myocardial infarction. The cardioprotective agent(s) on astent introduced into an individual is/are released from the stentwithin 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours,36 hours, 48 hours, or 72 hours after the stent is introduced into anindividual. Release of the cardioprotective agent(s) begins soon afterthe stent is introduced and typically within an hour after stentintroduction.

The cardioprotective agent(s) are released from the stent into themyocardial circulation at a concentration of about 0.01, about 0.1,about 1, about 10, about 100, or about 1000 pg/ml or any concentrationin between. In the case of IGF-1, a therapeutic dose of from 250 to 750pg is delivered into the myocardial circulation over a suitable periodof time, generally up to 14 days.

In the methods described herein, TGFβ1, IGF1, or TGFβ1 and IGF1 can beadministered in combination with other cardioprotective agents, such asother (different) cytokines, anti-coagulating agents, a vesselspasticity minimizing agents, a vasodilator agent, a calcium blockeragent, a sodium channel blocker agents, or an anti-inflammatory agents.

In another embodiment of the method of treating an individual sufferingfrom an acute myocardial infarction, a therapeutically effective amountof a (at least one, one or more) cardioprotective agent is administeredintravenously to the individual, upstream of the myocardial infarct. Inspecific embodiments, TGFβ1, IGF1, or TGFβ1 and IGF1 are administeredintravenously to the individual in sufficient quantity to result indelivery of the cardioprotective agent to the heart in an amount orconcentration sufficient to assist or enhance repair of heart tissue andreduce (completely or partially) apoptosis of cardiomyocytes and, thus,in particular, stimulate survival or repair of damaged cardiac muscle orstimulate left ventricular remodeling. In this embodiment, acardioprotective agent(s) is administered intravenously at the time ofmyocardial infarction (immediately) or within a few minutes to a fewhours, or within up to 72 hours after it has occurred. The dose ofTGFβ1, IGF1, or TGFβ1 and IGF1 administered in this embodiment isapproximately up to 5-fold higher, or 10-fold higher, or 50-fold higher,or 100-fold higher, or 500-fold higher, or up to 1000-fold higher thanthe dose administered into a coronary artery (e.g., than the doseadministered via stent introduced into a coronary artery). In thisembodiment as well, TGFβ1, IGF1, or TGFβ1 and IGF1 can be administeredin combination with additional cardioprotective agents, such as other(different) cytokines, anti-coagulating agent, a vessel spasticityminimizing agent, a vasodilator agent, a calcium blocker agent, a sodiumchannel blocker agent, or an anti-inflammatory agent.

In addition, provided herein are drug eluting stents that can beintroduced at the site of an acute coronary artery occlusion upstream ofthe site of acute myocardial infarction in order to treat an individualsuffering from acute myocardial infarction. The drug eluting stentsprovided deliver a (at least one, one) cardioprotective factor, such asTGFβ1, IGF1, or TGFβ1 and IGF1, when present in (after being introducedinto) the individual at the site and at the time of myocardialinfarction or shortly thereafter (e.g., within 5 minutes, 10 minutes, 15minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, or 72 hours).The drug eluting stent of the invention not only serves to maintainvessel patency after angioplasty, but also acts as a regional drugdelivery platform to enhance repair and reduce (partially or completely)apoptosis in the myocardium, such as in the infarct border zone.

The drug eluting stent of the invention, when surgically inserted, forexample, at the site of an acute coronary artery occlusions upstream ofthe site of acute myocardial infarction, releases cardioprotectiveagent(s) that exert certain effects, such as anti-apoptotic and/orcardiotrophic effects, that are beneficial to an individual sufferingfrom an acute myocardial infarction. The beneficial effects exerted bythe cardioprotective agents of the invention, when provided sufficientlysoon after the occurrence of myocardial infarction include a reductionor prevention of necrosis or apoptosis of myocytes, such as at theperiphery of developing myocardial infarcts and/or an induction ofgrowth of surviving myocytes. The cardioprotective agents disclosedherein may also exert their effects on cells other than myocytes. Theseeffects may lead to a reduction in size and/or partial or completerestoration of moderately injured and/or necrotic tissue of the affectedarea.

The drug eluting stent can be coated with single cardioprotective agentor combinations thereof, as well as with additional agents, such as, forexample, anti-coagulating agents.

Provided herein are methods for treating an individual having (sufferingfrom) an acute myocardial infarction and drug eluting stents useful fortreating such individuals. These methods include treating an individualby introducing, such as by surgically inserting, at a site of an acutecoronary artery occlusion upstream of the site of acute myocardialinfarction, a drug eluting stent (DES) that comprises a cardioprotectiveagent that is released from the stent in a therapeutically effectiveamount. A therapeutically effective amount of a cardioprotective agentis an amount that, when administered to an individual as describedherein, results in delivery of the cardioprotective agent to (presenceof the cardioprotective agent in) the heart in an amount orconcentration sufficient to assist or enhance repair of heart tissueand/or reduce (completely or partially) apoptosis of cardiomyocytes and,thus, reduce adverse effects on the heart.

Also provided herein are drug eluting stents that can be inserted at thesite of an acute coronary artery occlusion upstream of the site of acutemyocardial infarction. The drug eluting stent delivers a (at least one,one or more) cardioprotective agent, such as a tissue repair factor(e.g., cellular factors TGFβ1 and/or IGF1) to an individual after it isinserted into an individual. The drug eluting stent, in one embodiment,acts as an agent or drug delivery platform to enhance repair and/orreduce (partially or totally) apoptosis of myocytes, such as in theinfarct border zone, and/or to maintain vessel patency afterangioplasty.

Individuals who benefit from the method and stent described herein arehumans who are suffering from or experiencing a cardiovascular event,such as acute myocardial infarction (AMI). Acute myocardial infarctionis commonly known as a “heart attack.”

The term “myocardial infarction” relates to changes in the heart muscle(myocardium) that occur due to the sudden deprivation of circulatingblood, caused by events such as arteriosclerosis (narrowing or cloggingof the coronary arteries) and thrombosis (clot), which reduce the flowof oxygen to the heart. The main change is death (necrosis) ofmyocardial tissue, which can lead to permanent damage or death of theheart muscle.

In one embodiment the invention provides methods for treating anindividual having an acute myocardial infarction. These methods includetreatment of an individual by surgically inserting an agent or drugeluting stent at a site of an acute coronary artery occlusion upstreamof the site of acute myocardial infarction, for example during the PCIor coronary angiography procedures described herein.

In one embodiment, the eluting factor on the stent comprises acardioprotective agent, which is released from the stent after it isintroduced into an individual in need of treatment of myocardialinfarction. In one embodiment the cardioprotective agent exerts itscardioprotective effects near or at the infarct border zone (a site ofmoderately injured, partially perfused, potentially salvageable tissue,comprising myocytes, at the periphery of developing myocardialinfarcts).

In one embodiment the drug eluting stent is inserted at the time of theacute myocardial infarction, such as during the PCI or coronaryangiography procedures described herein. The methods as described hereincomprise inserting a drug eluting stent that comprises an eluting factorcomprising a cardioprotective agent and may be carried out within 5minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36hours, 48 hours, or 72 hours after the acute myocardial infarction.

In one embodiment methods described herein further comprise adjuvanttherapy, or may be combined with any form of diagnosis (e.g. history,ECG, and cardiac markers) and/or treatment (prophylactic or stabilizingtreatment, such as administering oxygen, aspirin, sublingual glyceryltrinitrate, and/or pain relievers, as well as treatment with betablockers, anticoagulation agents, ACE inhibitors, and/or antiplatelet)prior to stenting and also treatment after stenting (e.g., treatmentwith anti-lipemic agents, anti-inflammatory agents, anti-thromboticagents, fibrinolytic agents, anti-platelet agents, direct thrombininhibitors, glycoprotein IIb/IIIa receptor inhibitors, agents that bindto cellular adhesion molecules and inhibit the ability of white bloodcells to attach to such molecules (e.g. anti-cellular adhesion moleculeantibodies), alpha-adrenergic blockers, beta-adrenergic blockers,cyclooxygenase-2 inhibitors, angiotensin system inhibitor,anti-arrhythmics, calcium channel blockers, diuretics, inotropic agents,vasodilators, vasopressors, thiazolidinediones, cannabinoid-1 receptorblockers and/or any combinations thereof).

Stents are commonly used during angioplasty and other revascularizationprocedures. One embodiment of a stent is a small, coiled wire-mesh tubethat can be inserted into a blood vessel, such as an artery in theheart, that may be used to open a narrowed or clotted blood vessel.Stents may be permanently inserted into the artery during angioplasty.The stent may be expanded using a small balloon during the angioplastyprocedure. When the balloon inside the stent is inflated, the stentexpands and presses against the walls of the artery, which traps any fatand calcium buildup against the walls of the artery, allows blood toflow through the artery, and helps prevent the artery from closing again(restenosis). The stent may also help prevent small pieces of plaquefrom breaking off and causing a heart attack. Mesh-like stents allowblood vessel cells to grow through the mesh lining the stent and helpingto secure it. The balloon is then deflated and removed, leaving thestent in place. Balloon angioplasty is often used to insert stents,although sometimes stents are placed without the use of a balloon, andother methods are known to those in the art.

Stents are provided that comprise a drug or agent that is released fromthe stent at a controlled rate and concentration over a specified timeinterval upon insertion, e.g. at a site of an acute coronary arteryocclusion upstream of the site of acute myocardial infarction. Thesedrug-eluting stents are stents that are coated with agents. These agentsmay be cardioprotective agents or other agents. Drug-eluting stents areknown in the art and are described for example in U.S. Pat. Nos.5,591,227, 5,697,967, 5,599,352, U.S. Publ. No. 2007/0077266, and PCTapplications WO 01/12779, and WO 90/13332.

The invention provides, in one embodiment, drug eluting stents thatdeliver one or more cardioprotective agents, such as tissue repairfactors (e.g., cellular factors TGFβ1 and IGF1). In one embodiment, thestent is coated with an eluting factor, from which the one or morecardioprotective agents is released after the stent is introduced intoan individual. In other embodiments, the drug eluting stent releasescardioprotective agents and one or more additional agents (other thanTGFβ1 and IGF1).

IGF1 eluting stents are known in the art for delivery of micromolar ornanomolar amounts of IGF1 into the coronary circulation, examplesinclude: U.S. Pat. No. 6,660,034 covers the release of IGF1 and IGF2from stents with sole objective of enhancing angiogenesis: U.S. patentSer. No. 10/678,763 claims the release of Insulin like growth factorsfrom stents with the objective of enhancing blood flow, it does notspecify the IGF isoform or provide data on the dose, it is, focused onangiogenesis: U.S. Pat. No. 7,491,234 teaches the release of IGF1 overpolymer coated vascular stent but does not indicate the therapeuticinterest; U.S. Pat. No. 7,361,339 teaches the anti-apoptosis effects ofIGF in acute MI but doesn't provide any data on the dose or the IGF1release kinetics; U.S. Pat. No. 7,357,940 claims the release of IGF overpolymer coated vascular stent but does not indicate the dose or the IGFrelease kinetics; U.S. Pat. No. 7,351,421 claims the release of IGF overpolymer coated vascular stent, but does not indicate the dose or the IGFrelease kinetics: U.S. Pat. No. 7,332,160 claims the release of IGF overpolymer coated vascular stent: U.S. Pat. No. 7,252,818 teaches IGF1 andIGF 2 for angiogenesis; U.S. Pat. No. 7,241,455 claims the release ofIGF over polymer coated vascular stent; U.S. Pat. No. 7,101,857 claimsthe release of IGF over polymer coated vascular stent; U.S. Pat. No.7,055,237 claims the release of IGF over polymer coated vascular stent;U.S. Pat. No. 7,022,132 claims the release of IGF over polymer coatedvascular stent; U.S. Pat. No. 6,923,996 claims the release of IGF overpolymer coated vascular stent; U.S. Pat. No. 6,830,577 claims therelease of IGF over polymer coated vascular stent; U.S. Pat. No.6,720,141 claims the release of IGF over polymer coated vascular stentbut is focused at In-stent restenosis; U.S. Pat. No. 6,709,427 claimsthe release of IGF over polymer coated vascular stent; U.S. Pat. No.6,613,084 claims the release of IGF over polymer coated vascular stent:U.S. Pat. No. 6,569,195 claims the release of IGF over to polymer coatedvascular stent; U.S. Pat. No. 6,569,147 claims the release of IGF overpolymer coated vascular stent; U.S. Pat. No. 6,258,121 claims therelease of IGF over polymer coated vascular stent; U.S. Pat. No.5,954,706 claims the release of IGF over polymer coated vascular stent;U.S. Pat. No. 6,379,382 teaches multiple drug coating on the stents andclaims the release of IGF. The contents of all of these applications areincorporated herein by reference.

The cardioprotective agents and other agents, alone or in combination,can be combined with organic or inorganic carrier molecules, elutionfactors, solvents, salts, biopolymers, synthetic polymers and applied tothe stent to generate a coated stent. Stent coating is known in the artand may involve immersion of the stent in a solution or may involvespray coating.

Drug-eluting stents may be coated with cells, such as endothelial cellsengineered to express cellular factors that have, for example,cardioprotective, angiogenic, anti-thrombotic, antiplatelet,anticoagulant, antimicrobial, anti-inflammatory, antimetabolic, and/orvasoreactive effects, or may be directly coated with genes encodingpolypeptides exerting similar effects. Drug-eluting stents may be coatedwith agents such as cardioprotective agents, angiogenic agents,anti-thrombotic agents, antiplatelet agents, anticoagulant agents,antimicrobial agents, anti-inflammatory agents, antimetabolic agents,and/or vasoreactive agents.

An anti-inflammatory factor or agent is, for example, a cytokine withinflammation inhibitory action, such as TGF-β, IL-4, IL-5, IL-10, IL-13,galectin-3. An angiogenic factor is for example a cytokine transmittinga signal for stimulation of angiogenesis, such as HGF, VEGF, bFGF, TNF-αor TP.

Agents that may be used for coating of drug-eluting stents may bederived from bioartificial polymeric materials obtained from blends ofsynthetic polymers with, for example, fibrin, thrombin and/or collagen,wherein for example a synthetic or biodegradable polymer is combined,for example through mixing, with fibrinogen and cross-linked withthrombin and then made into vascular grafts.

Suitable biostable and/or synthetic polymers include silicones,polyurethanes, polyesters, vinyl homopolymers and copolymers, acrylatehomopolymers and copolymers, polyethers and cellulosics. Suitablebioabsorbable and/or biodegradable polymers include polyphosphate ester,polyhydroxybutyrate valerate, polyhydroxybutyrate-co-hydroxyvalerate,poly(L-lactic acid), poly(lactide-co-glycolide) andpoly(hydroxybutyrate-co-valerate).

Drug-eluting stents can be generated by various methods known in theart. One such method comprises making or obtaining a solution whichincludes a solvent, a polymer dissolved in the solvent and a therapeuticagent, such as a cardioprotective agent, anti-thrombotic agent,antiplatelet agent, anticoagulant agent, antimicrobial agent,anti-inflammatory agent, antimetabolic agent, and/or vasoreactive agentas described herein, dispersed in the solvent to obtain a solution andapplying the solution to the structural elements of a stent by immersingthe stent into the solution or by spraying the solution onto the stentand evaporating the solution.

Any combinations of spreading, dipping or spraying using water and/or anorganic solvent capable of dissolution of the agent and the bindercomponent, such as a polymer followed by drying (natural drying ordrying under reduced pressure or the like) may be suitable forgenerating a drug eluting stent, and such methods are known in the art.The inclusion of a polymer in intimate contact with a therapeutic agentor drug on the underlying stent structure allows the therapeutic agentor drug to be retained on the stent in a resilient matrix duringexpansion of the stent and also slows the administration of drugfollowing implantation. The method can be applied whether the stent hasa metallic or polymeric surface. The amount of drug to be included onthe stent can be readily controlled by applying multiple thin coats ofthe solution while allowing it to dry between coats. The overall coatingshould be thin enough so that it will not significantly increase theprofile of the stent for intravascular delivery by catheter, such as forexample, less than about 0.002 inch thick or less than 0.001 inch thick.The adhesion of the coating and the rate at which the drug is deliveredcan be controlled by the selection of an appropriate bioabsorbable orbiostable polymer and by the ratio of drug to polymer in the solution.By this method, therapeutic agents or drugs such as glucocorticoids(e.g. dexamethasone, betamethasone), heparin, hirudin, tocopherol,angiopeptin, aspirin, ACE inhibitors, growth factors, oligonucleotides,and, more generally, cardioprotective agents, antiplatelet agents,anticoagulant agents, antimitotic agents, antioxidants, antimetaboliteagents, and anti-inflammatory agents can be applied to a stent, retainedon a stent during expansion of the stent and elute the drug at acontrolled rate. The release rate can be further controlled by varyingthe ratio of therapeutic agent or drug to polymer in the multiplelayers.

For example, a higher drug-to-polymer ratio in the outer layers than inthe inner layers would result in a higher early dose, which woulddecrease over time. A typical ratio of drug to dissolved polymer in thesolution can vary widely (e.g. in the range of about 10:1 to 1:100). Inone embodiment, the drug eluting stent provided comprises an elutingfactor that comprises a (at least one, one or more) cardioprotectiveagent and is capable of releasing/releases the cardioprotective agentunder physiological conditions/when the stent is present in the site ofan acute coronary artery occlusion upstream of the site of acutemyocardial infarction. In certain embodiments, the cardioprotectiveagent is released from the stent (from the eluting factor) within 5minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2hours, 3 hours, 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36hours, 48 hours, or 72 hours after the stent is inserted into theindividual. In another embodiment, the cardioprotective agent isreleased from the stent at a concentration of 0.01, 0.1, 1, 10, 100, or1000 pg/ml or any concentration in between after the stent is inserted.The time after introduction of the stent into an individual at whichrelease of the cardioprotective agent begins and the concentration orrate at which the cardioprotective agent is released can be varied asneeded to provide a therapeutically effective amount of thecardioprotective agent in an appropriate time period (in sufficient timeto have the desired effect of assisting or enhancing repair of hearttissue and/or reducing (completely or partially) apoptosis ofcardiomyocytes and, thus, reducing adverse effects on the heart). Thetime of release after introduction of the stent into an individual andthe concentration or rate at which the cardioprotective agent isreleased can be combined in any manner (e.g., release in a short time ata low concentration, fast release at a high concentration), in order toprovide effective treatment. Further, a stent can comprise one or morecardioprotective agents (e.g., TGFβ1 alone or in combination with IGF1and/or another cardioprotective agent: IGF1 alone or in combination withTGFβ1 and/or another cardioprotective agent). It can further compriseother agents (drugs), such as those that have anti-apoptotic effectsand/or cardiotrophic effects, tissue repair factors, cytotoxic agent(s),cytostatic agent(s) or anti-coagulating agent(s).

As used herein, the term “cardioprotective agents” refers to agents thatexert certain effects, such as anti-apoptotic and/or hypertrophiceffects, that are beneficial to the patient or subject suffering from anacute myocardial infarction. Cardioprotective agents of the inventionmay exert their beneficial effects on cells such as for example myocytesin tissues such as heart tissues, but may also exert their effects oncells such as epithelial or endothelial cells present for example inarteries supplying the heart. When supplied sufficiently early after theoccurrence of myocardial infarction the beneficial effects may include areduction or prevention of cell death, such as through necrosis orapoptosis, of myocytes at the periphery of developing myocardialinfarcts and/or an induction of growth of surviving myocytes. Thecardioprotective agents may also stimulate growth or prevent cell deathof epithelial or endothelial cells present in arteries supplying theheart. These effects may lead to a reduction in size and/or partial orcomplete restoration of the moderately injured and/or necrotic tissue ofthe infarcted area. Additional beneficial effects may include dissolvingblood clots or other organic material leading to arterial constrictionsor occlusions, such as vulnerable atherosclerotic plaques.

Cardioprotective agents disclosed herein that exert anti-apoptoticand/or hypertrophic effects include TGFβ1 and IGF1. Other cellularfactors that are involved in tissue repair or maintenance, stem cellfactors, anti-apoptotic factors and/or growth factors may also be usefulas cardioprotective agents. Such agents include granulocyte colonystimulating factor (GCSF), platelet-derived growth factor (PDGF),vascular endothelial growth factor (VEGF), fibroblast growth factors(FGFs), Angiotensin II, and stromal cell-derived factor 1 (SDF-I).

TGF-β is a secreted protein that exists in three isoforms TGF-β1, TGF-β2and TGF-β3. The TGF-β family is part of a superfamily of proteins knownas the transforming growth factor beta superfamily, which includesinhibins, activin, anti-müllerian hormone, bone morphogenetic protein,decapentaplegic and Vg-I.

The insulin-like growth factors (IGFs) are polypeptides with highsequence similarity to insulin and comprise of cell-surface receptors(e.g. IGF1R and IGF2R), ligands (e.g. IGF-1and IGF-2), IGF bindingproteins (e.g. IGFBP 1-6), as well as associated IGFBP proteases.Insulin-like growth factor 1 (IGF-1) is mainly secreted by the liver asa result of stimulation by growth hormone (GH) and plays a role in thepromotion of cell proliferation and the inhibition of cell death(apoptosis). Insulin-like growth factor 2 (IGF-2) is thought to be aprimary growth factor required for early development while IGF-1expression is required for achieving maximal growth.

Many cardioprotective agents disclosed herein, such as TGFβ1 and IGF1,are commercially available in purified or recombinant form and have beensuggested for many therapeutic uses, such as for example wound healing(U.S. Pat. Nos. 4,861,757; 4,983,581 and 5,256,644), or induction ofbone growth (U.S. Pat. Nos. 5,409,896 and 5,604,204).

The cardioprotective agents disclosed herein can also be combined withadditional agents that have complementary, synergistic or additiveeffects. For example, drugs that lower cholesterol levels, such asstatins, may enhance the responsiveness of cardiovascular cells to theprotective actions of TGF-β, thus helping prevent the development ofatherosclerosis and heart disease. In another examples, anti-coagulatingagents such as Aspirin (salicylic acid), heparin, Coumadin,ethylenediamine tetraacetic acid (EDTA), citrate,ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA),diethylenetriamine pentaacetic acid (DTPA), 1,2-diaminocyclohexanetetraacetic acid (DCTA) and others, may be combined withcardioprotective agents to help prevent arterial blockage. Vesselspasticity minimizing agents, such as haloalkylamine alpha adrenergicblocking agents, e.g. phenoxybenzamine, isomers of phenoxybenzamine andtertiary amines of phenoxybenzamine, and/or vasodilator agents, such aslidocaine, xylocaine, tetracaine, procaine and other short-termvasodilators such as papaverine, adenosine, nitric oxide donor agents,calcium channel blocker agents, sodium channel blocker agents andrelated adenosine regulating agents may also be combined withcardioprotective agents. Anti-inflammatory agents, such as non-steroidalanti-inflammatory drugs (NSAIDs), for example ibuprofen, aspirin andnaproxen, or herbs with anti-inflammatory effects, such as hyssop,ginger, Turmeric, Arnica montana and willow bark may also be combinedwith cardioprotective agents.

Cardioprotective agents of the invention may also be combined withadditional cardiovascular agents, such as aldosterone receptorantagonists, angiotensin converting enzyme inhibitors, angiotensin IIinhibitors, centrally and peripherally acting antiadrenergic agents,antiadrenergic agents, antianginal agents, antiarrhythmic agents,beta-adrenergic blocking agents, cardioselective and non-selective betablockers, calcium channel blocking agents, diuretics (e.g. loop,potassium-sparing, thiazide diuretics), carbonic anhydrase inhibitors,inotropic agents, vasodilators, renin inhibitors, sclerosing agents, andvasopressin antagonists.

Examples of alpha-adrenergic blockers include: doxazocin, prazocin,tamsulosin, and tarazosin.

Beta-adrenergic receptor blocking agents are a class of drugs thatantagonize the cardiovascular effects of catecholamines in anginapectoris, hypertension, and cardiac arrhythmias. Beta-adrenergicreceptor blockers include, but are not limited to, atenolol, acebutolol,alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol,hedroxalol, indenolol, labetalol, levobunolol, mepindolol, methypranol,metindol, metoprolol, metrizoranolol, oxprenolol, pindolol, propranolol,practolol, practolol, sotalolnadolol, tiprenolol, tomalolol, timolol,bupranolol, penbutolol, trimepranol,2-(3˜(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl,1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)˜2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy) phthalide. The above-identifiedcompounds can be used as isomeric mixtures, or in their respectivelevorotating or dextrorotating form.

Patients without ST segment elevation are presumed to be experiencingeither unstable angina (UA) or non-ST segment elevation myocardialinfarction (NSTEMI). NSTEMI may be managed with medication (e.g.antiplatelet and anticoagulating agents) to prevent the narrowed arteryfrom occluding, although PCI is often performed during hospitaladmission. In patients who have multiple blockages and who arerelatively stable, bypass surgery of the blocked coronary artery may bean option. Coronary artery bypass surgery involves an artery or veinfrom the patient being implanted to bypass narrowing or occlusions onthe coronary arteries. Several arteries and veins can be used, howeverinternal mammary artery grafts have demonstrated significantly betterlong-term patency rates than great saphenous vein grafts.

Post-MI, several long-term medications can be used with the aim ofpreventing secondary cardiovascular events such as further myocardialinfarctions, congestive heart failure or cerebrovascular accident (CVA).Antiplatelet drug therapy such as aspirin and/or clopidogrel, Betablocker therapy such as metoprolol or carvedilol, ACE inhibitor therapy,Statin therapy, aldosterone antagonist agents, such as eplerenone,Omega-3 fatty acids, and/or stem cell treatment by coronary arteryinjections of stem cells derived from their the patient's own bonemarrow, may be used.

Therapies for reducing the risk of a future cardiovascular event includebut are not limited to diet and/or exercise and/or therapies with:anti-lipemic agents, anti-inflammatory agents, anti-thrombotic agents,fibrinolytic agents, anti-platelet agents, direct thrombin inhibitors,glycoprotein IIb/IIIa receptor inhibitors, agents that bind to cellularadhesion molecules and inhibit the ability of white blood cells toattach to such molecules (e.g. anti-cellular adhesion moleculeantibodies), alpha-adrenergic blockers, beta-adrenergic blockers,cyclooxygenase-2 inhibitors, angiotensin system inhibitor,anti-arrhythmics, calcium channel blockers, diuretics, inotropic agents,vasodilators, vasopressors, thiazolidinediones, cannabinoid-1 receptorblockers and/or any combinations thereof.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thepreceding following description or illustrated in the drawings. Theinvention is capable of other embodiments and of being practiced or ofbeing carried out in various ways. Also, the phraseology and terminologyused herein is for the purpose of description and should not be regardedas limiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

EXAMPLES

Cardiotrophic effects of conditioned media (CM) derived from autologouscirculating progenitor cells (CPCs) in a porcine myocardial infarction(MI) model were investigated and the cellular factors TGFβ1 and IGF1were implicated to play a role in the observed beneficial effects.Landrace pigs (25-28 Kg) underwent MI generation via percutaneoustransluminal balloon occlusion of the coronary artery for 80 minutes,followed by 120 minutes of reperfusion. Intra-coronary conditioned media(CM) from autologous CPC or CM+anti-TGFβ1 or CM+anti-IGF1 or X-vivo 15(control) was administered during balloon occlusion of the infarctrelated artery. At 24 hours or 8 weeks post MI, the animals weresacrificed, and the hearts underwent infarct quantification.Cardiomyocyte apoptosis within the MI borderzone (50% MI/50% normalmyocardium/high power field) was evaluated by TUNEL staining, Caspase 9and BCl-2 protein expression together with Caspase 3 and 9 activityassay. Functional analysis was performed using conductancepressure-volume catheters. CM therapy significantly reduced the infractsize at 8 wks. In addition CM significantly reduced the apoptotic signalat 24 hours, and a significant improvement in cardiac function wasobserved at 24 hrs as well as 8 weeks post MI. The beneficial effects ofCM were attenuated by blocking TGFβ1 and IGF1. Thus, CPC derivedconditioned media reduces cardiomyocyte apoptosis in the MI borderzone24 hours post MI generation, leading to an improvement in leftventricular function. These beneficial effects of CM are mediatedsubstantially through TGFβ1 and IGF1.

Materials and Methods

Isolation and Characterization Circulating Progenitor Cells fromPeripheral Blood:

Circulating progenitor cells (CPCs) were isolated from 30-40 mIs ofblood draw-n from an ear vein of female Landrace pigs under aseptictechnique 48 hours before infarct generation, as previously described(Doyle BJ. et al., Stem Cells Dev. 2008 Jun. 18). Blood mononuclearcells were harvested from peripheral buffy coat preparations usingFicoll-Paque Plus (15 cc) separation. The cells were analysed for theexpression of CPC markers (CD133, CD34, cKit, FLK1, VE-Cadherin andeNOS) by RT PCR and were compared to human CPCs. Total RNA was isolatedfrom 24 hour cultured human and pig CPC using SV Total RNA Isolation kit(Progenia, Southampton, UK). 5 μg of total RNA was reverse transcribedusing Superscript III First-Strand Synthesis Kit with poly-dT primers(Invitrogen, Carlsbad, Calif.). 2 μl of cDNA was used for each PCRreaction for stem/endothelial cell markers using Platium Taq Polymerase(Invitrogen, Carlsbad, Calif.) with the primers and annealingtemperatures outline in Table 1 below. Amplicons were visualized on 1.5%agarose gel stained with SybrGreen I nucleic acid stain (Invitrogen,Carlsbad, Calif.).

TABLE 1 The following primer sequences and RT PCR conditions were used:Marker/ Size of Anneling Sequence Primer Sequence Product Tm (° C.)ID NO CD133 For 5′-TCCTGGGGTTGCTGTTTATT-3′ 157 bp 58    1 Rev5′-CATATCACCAAGAGGGAAACG-3′  2 cKit For 5′-GAGGAGATAAATGGAAAC-3′ 385 bp51    3 Hum-Rev 5′-GAATCACGTTTTCTTCTC-3′  4 Pig-Rev5′-GAATCACGTTTCCGTCTC-3′5′  5 CD34 Hum-F 5′-CCTGATGAATCGCCGCAGCTGGAGC-3′201 bp 58    6 Hum-R 5′-CCAGAAACGGCCATTCAGCAAGACA-3′  7 Pig-F5′-GCTGATGAACCGTCGCAGTTGGAGC-3′  8 Pig-R 5′-CCAGAAACGGCCATTCAGCGAGGCA-3′ 9 Flk-1 For TTATCGGAGAAGAACGTGGT 412 bp 55   10 RevTAATGCTCAGCAGGATGGCA 11 eNOS For 5′-GGTATGGATGAGTATGACGTG-3′ 171 bp 51  12 Rev 5′-TGTTCCGGCCGAGGG-3′ 13 VE- For 5′-AACTTCCCCTTCTTCACCC-3′ 368 bp51   14 Cadherin Rev 5′-AAGGCTGCTGGAAAATG-3′ 15 G3PDH For5′-CCATGTCGTCATGGGTGTGAACCA-3′ 251 bp 59.9 16 Rev5′-GCCAGTAGAGGCAGGGATGATGTTC-3′ 17

Generation of Conditioned Media from Circulating Progenitor Cells:

To generate the conditioned media, the CPCs were washed three times inMCDB 131 supplemented with hydrocortisone, antibiotics, and 10 ng/mlVEGF. Following this step, cells were subsequently re-suspended inX-Vivo-15 medium (BioWhittaker) supplemented with VEGF (1 ng/ml), andseeded on fibronectin coated plates at a density of 4.9×10³ cells permm² in a modification of previously reported methodology (Assmus B. etal., Circulation. 2002; 106: 3009-17; Doyle BJ. et al., Stem Cells Dev.2008 Jun. 18). After 48 hours of culture, conditioned cell-free mediawas harvested from the cell culture by centrifugation (600×g for 5minutes) and filtration (0.2 μm) of the media. The condition media wasscreened for presence of cytokines using the RayBio Human CytokineAntibody Array kit.

In-Vitro Screening for Anti-Apoptotic Effects of Conditioned Media UsingNeonatal Cardiomyocytes:

Hearts were removed from 1 day old Fisher rats, and cardiomyocytes wereisolated and cultured as previously described (Doyle BJ. et al., StemCells Dev. 2008 Jun. 18; Perez-Terzic C. et al., Circ Res. 1999; 84:1292-301). In line with a pure cardiomyocyte preparation, these cellsshowed no evidence of neural or vascular cell contamination.Cardiomyocytes were incubated under hypoxic conditions (95% CO₂ 5% O₂)for 24 hours at 37° C., followed by normoxic condition for 24 hours at37° C. to induce cell death by apoptosis. Before the induction ofhypoxia the cardiomyocytes were treated with the following solutions:fresh media (X vivo-15 with 1 ng/ml VEGF), conditioned media (CM)obtained from porcine CPC cultures (as described above), conditionedmedia containing a 100-fold excess of neutralizing antibody to eitherTGFβ or IGF1 or IgG₅ and Purified porcine TGFβ (10-1000 pg/mL) or IGF1(1-100 pg/mL). The apoptosis signal in the cardiomyocytes was quantifiedusing commercial caspase 9 activity assay kit (Chemicon International,California, USA) and the data is expressed as percent of X-Vivo treatedgroup.

Porcine Model of Myocardial Infarction:

39 female Landrace pigs weighing 25-30 kg were used in this study inaccordance with the guidelines of Experimental Animal Ethics Committeeof University College Cork. 20 out bred farm reared juvenile Landracepigs underwent percutaneous balloon occlusion of the proximal leftcircumflex (LCx) coronary artery for 80 minutes. The chronic infarctgroup comprising 19 similar pigs had percutaneous proximal left anteriordescending (LAD) coronary artery balloon occlusion for an identicalballoon inflation period. This experimental protocol has been describedin detail previously (Klein HH. et al., Basic Res Cardiol. 1984; 79:440-7) Briefly all animals were pre-medicated with amiodarone 400 mg,aspirin 75 mg and clopidogrel 75 mg daily for 8-10 days prior to infarctgeneration. Following intramuscular injection of ketamine and xylazine,the animals received intravenous propofol to effect. Mechanicalventilation was carried out using a large animal Harvard Apparatusventilator and supplemental oxygen (4-6 L/min) combined with isoflurane(1-4%) to maintain general anaesthesia. Under fluoroscopy a 3.0×13 mm(Boston Scientific, Galway, Ireland) angioplasty balloon was inserted inthe proximal LCx (acute infarct group) or the proximal LAD (chronicgroup) using a 6¥ IMA guide catheter (Boston Scientific, Galway Ireland)introduced via a 7Fr arterial sheath from the right internal carotidartery. The balloon was inflated to 6-8 atm, ensuring completeangiographic occlusion of the target vessel with check angiographyperformed every 15-20 minutes during the infarct generation period.Significant ST-segment elevation was confirmed on electrocardiography inall cases. Malignant arrhythmias were defibrillated as necessary. Theballoon was then deflated and removed to allow reperfusion for a totalof 120 minutes. Check angiography confirmed TIMI-3 flow in the infarctrelated artery at initiation of reperfusion. An over-the-wire coronaryballoon (3.0×12 mm. Boston Scientific Galway. Ireland) was thenpositioned at the site of prior vessel occlusion. Three cycles of 4minute balloon inflation with intracoronary infusion of 4 ml ofX-vivo/VEGF, conditioned media. CM+anti-IGF 1, CM+anti-TGFβ1, or CM+IgGwas performed, with a period of 4 minutes of balloon deflation betweeneach cycle. For the CM+anti-IGF1, CM+anti-TGFβ1, or CM+IgG group, theblocking antibody or the IgG was mixed in CM 15 mins prior tointracoronary delivery. Finally check angiography confirmed infarctrelated artery patency (TIMI-3 flow), and the animal was recovered.

24 hours (acute study) or 8 weeks (chronic study) after infarctgeneration, the animals underwent repeat coronary angiography to confirminfarct related artery TIMI-3 patency, via the left internal carotidartery as described above. Following completion of recording ofhaemodynamic parameters with the conductance catheter, the animals weresacrificed with an overdose of pentobarbitone. The hearts wereexplanted, weighed and sectioned in 5 mm transverse slices from apex tobase (6-8 slices/heart). A representative mid ventricular slice (3^(rd)or 4^(th) slice) was stained with 2% TTC. Images of the sections werecaptured using a 10 mega pixel digital camera. Infarct area wasquantified with planimetry of the images using NIH Image J software(Maryland, USA). Myocardium was taken from infarct, borderzone andremote myocardial areas for OCT embedding and cryopreservation in liquidnitrogen.

Pressure-Volume Loop Protocol:

Pressure volume loops were recorded using a 5Fr pig tailed conductancecatheter (Miller Instruments, Houstan Tex., USA) positioned in the leftventricular apex under fluoroscopic guidance prior to MI generation,during reperfusion, post treatment and immediately prior to sacrifice(24 hours-acute group, 8 weeks-chronic group). Recordings were taken inthe steady state in sinus rhythm for a minimum of 10 minutes using asample frequency of 250 Hz, using LabChart 5 Pro (AD InstrumentsOxfordshire, UK) and off-line analysis off haemodynamic parameters wasperformed using PVAN ultra 1.0 software (AD Instruments Oxfordshire,UK). A minimum of 45 sec of pressure-volume recordings in the steadystate was analysed per animal (n=3 per treatment group).

Histology

5 μM thick sections from OCT embedded infarct, border zone myocardiumwere cut for histological analysis. The sections were stained withhaematoxylin and eosin, Sirius red or Mason's Trichrome. Representativeslides from infarct borderzones of the chronic group (as defined above)were stained for collagen content with picrosirius red (Sirius red F3BAin aqueous picric acid). At least 36 high powered fields/treatment groupwere acquired using Nikon CCD camera attached to the Nikon microscope.Collagen content was quantified by expressing regions stained withpicrosirius red as a percentage of the total infarct-borderzone area perhigh power field, employing an automated image analysis system (NISElements Basic Research software).

Apoptosis in Infarct Border Zone:

The apoptosis signals from the border zone myocardium were quantifiedusing three different established methodologies namely TUNEL(immunofluorescence), protein expression of Caspase 9 and bcl-2 usingWestern blots and by measuring activity of caspase 3 and 9. The infarctborderzone was defined as a high power field composed of 50% normalmyocardium and 50% infarct area. The total number of TUNEL positivecells was counted in 25 high power fields per animal, derived from 4slides (Sum thickness) taken from 3 mid-ventricular slices using the InSitu Cell Death Detection Kit (Roche) according to the manufacturer'sinstructions. Nuclei were counter-stained with DAPI. The detection ofthe TUNEL positive signal within cardiomyocytes was validated by dualstaining of the myocardium with anti-α sarcomeric actin (Sigma A2172)followed by Rhodamine conjugated goat anti-mouse IgM as secondaryantibody (Chemicon AP 128R).

Protein Extraction and Western Blot:

Expression of caspase 9 and bcl-2 were evaluated by Western blotanalysis. Tissue of infarct border zone were washed in ice-cold PBS,placed in lysis buffer (3 times the weight of sample in volume; 50 mm/LNaCl, 50 mmol/L NaF, 50 mmol/L sodium pyrophosphate, 5 mmol/L EDTA, 5mmol/L EGTA, 2 mmol/L Na3VO4, 1% Triton X-100, 10 mmol/L HEPES;supplemented with Complete protease inhibitors [Roche]) and homogenisedon ice. Samples were incubated 1 hour on ice, and then cleared ofcellular debris by centrifugation (14,000 rpm; 10 min). Protein levelswere assessed using Bradford reagent. One hundred micrograms of proteinper sample was prepared in sample buffer and loaded on 12%, thentransferred onto nitrocellulose membranes. Membranes were blocked in 5%milk and primary antibodies were applied caspase 9 (Stressgen), Bcl-2(Abeam) over-night at 4° C. Membranes were washed and the appropriatesecondary peroxidase-conjugated antibody (Jackson laboratories) wasapplied for 1 hour at room temperature. The bound antibodies werevisualized by chemiluminescence (SuperSignal, Pierce). Bandscorresponding to the correct molecular weight were quantified usingImage J (NIH software).

Caspase 3 and 9 Activity Assay:

Protein extraction was performed as described above for Western blotsbut without the Complete protease inhibitors. The tissue lysate was usedfor the estimation of caspase 3 and 9 activity using the caspase 3 and 9activity assay kit (Chemicon International, California. U.S.). Proteincontent of the lysate was determined using the Bradford Proteinestimation method and the caspase 3 and 9 activities were expressed asUnits/mg protein.

Cardiomyocyte Hypertrophy Assessment:

The mean cardiomyocyte size of pigs in each experimental chronic groupwas evaluated from frozen sections of the infarct borderzone, usingestablished morphometric methodology (Doyle BJ. et al., Stem Cells Dev.2008 Jun. 18; Senthil V. et al, Circ Res. 2005; 97: 285-92). Briefly,slides were incubated with anti-laminin antibody (Sigma L8271) andanti-α sarcomeric actin (Sigma A2172) and subsequently labelled withAlexa Fluor 488 (Molecular Probes) and goat anti-mouse IgMRhodamine-conjugated antibody (Chemicon AP 128R). Cell nuclei werestained with DAPI. At least 3 distinct sections from the infarctborderzone in each animal, and a minimum 1000 cardiomyocytes werestudied in each treatment group. Cell size was quantified by measuringcell surface area with laser confocal microscopy (Nikon TE 2000) and a40× objective. Two-dimensional confocal images were acquired by scanning1024×1024 pixels per image, and processed with NIS Elements BasicResearch software.

Fabrication of IGF-1 Eluting Stent:

Programmable elution profile (PEP™) based on a biostable polymericcoating is employed in fabrication of the IGF1 eluting stent. Using thePEP™ technology, a bare metal stent surface is modified to optimise thesurface properties before a polymer coating containing the IGF1 isapplied. The polymer containing the entrapped drug(s) is then sprayedonto the primed stent. The PEP™ system uses two polymers such that‘programmed’ release can be achieved using different PEP™ polymercombinations. The system has flexibility in that it can releasewater-soluble and water-insoluble drugs with fast or slow release rates.It has also been shown to deliver high and low molecular weight drugs.

A bare metal stent is de-greased (by treatment with NaOH undersonication followed with rinsing with distilled water and oven drying)and primed by contact with an alkoxysilane in an aprotic organic solvent(Toluene) in the presence of an acid catalyst (glacial acetic acid)resulting in the formation of covalent bonds. The treated stent is driedin high temperature (50-55° c.) under vacuum. The purpose of thispriming step is to create a monolayer. A bridging polymer is then addedon to this to enable attachment of biologically active agent such asIGF1. Carboxymethyl cellulose (CMC; molecular weight 5000-500,000),dextran or diisocyanate or combinations of these polymers are suitablefor this purpose. The strength of the polymer is inversely proportionalto the release of the biological agent. For a 24 hr IGF1 eluting stentIGF1 will be added to the polymer solution (0.025% CMC, 1 mole of adiamine and two moles of a diisocyanate group) to make finalconcentrations of 500-550 pg. This polymer/drug solution is sprayed intothe stent surface from a piezoelectric nozzle with a 40 um orificepositioned by a micromanipulator. The solvent is evaporated in 30minutes to leave a polymer/IGF 1 layer inside the stent. Processingvariables, such as polymer molecular weight, viscosity, number oflayers, layer thickness, drying time, temperature, humidity, and solventtype can influence kinetics and thus should be tightly controlled. Theloading process is repeated seven times to develop IGF1 eluting stentswith release kinetics over 7 days.

Example 1 Characterization of Circulating Progenitor Cells and CytokineArray of Condition Media

Circulating progenitor cells (CPCs) isolated from peripheral bloodmononuclear cells were characterized for expression of progenitormarkers (CD133, CD34, cKit, Flk1, VECadherin, eNOS) using RT-PCR withGAPDH as a control (FIG. 1A).

The presence of cytokines was measured in cell-free conditioned mediaobtained from CPCs after 48 hours of culture and harvesting bycentrifugation and filtration (0.2 μm). The conditioned media (CM) wasscreened for presence of cytokines using the RayBio Human CytokineAntibody Array kit (FIG. 1A, bottom right panel).

The CM from CPC was screened for presence of about 78 differentcytokines of which the TGFβ2 and IFG1 were the predominant growthfactors (as measured in percent of total cytokines, see FIG. 1B).

Example 2 Antiapoptotic Effects of Condition Media in NeonatalCardiomyocytes is Mediated by TGFβ and IGF1

Conditioned media significantly (about 50%) protected rat neonatalcardiomyocytes, isolated from hearts of 1 day old Fisher rats, fromapoptosis induced cell death, when incubated under hypoxic conditions.Apoptosis was reduced by about 50% as judged by caspase 9 activity.Blocking TGFβ and IGF1 using specific neutralizing antibodies abrogatedthe protective effects of conditioned media (FIG. 1C). For neutralizingof TGFβ and IGF1, the CM was incubated with a 100-fold excess ofanti-TGFβ and/or anti-IGF1 antibody for 15 minutes at 37° C. beforeexposing it to the cardiomyocytes. Purified TGFβ and IGF1 peptidesmimicked the effects of CM at a narrow dose range (FIG. 1D). The data isexpressed as Mean±SEM of % Caspase 9 activity to that of X-Vivo group inthree independently performed experiments.

Example 3 Conditioned Media Protects the Borderzone Myocardium fromApoptosis at 24 Hours Post Myocardial Infarction

The apoptosis signals from the border zone myocardium 24 hours post MIwere quantified using TUNEL (immunofluorescence), protein expression ofCaspase 9 and bcl-2 (Western blot) and by measuring activity of caspase3 and 9 (Caspase activity assay kit).

The infarct borderzone was defined as a high power field (200×) composedof 50% normal myocardium and 50% infarct area. Conditioned media (CM)treated group had significantly (p<0.001) reduced TUNEL signals at theborderzone myocardium compared to the X-Vivo treated group (FIG. 2A,compare upper panel with lower panel).

The effects of CM were blocked by neutralizing TGFβ and IGF 1 in CM(FIG. 2B). The TUNEL signals from 5 sections/pig (N=3-4) with an averageof 5 High power fields/section is expressed as Mean±SEM. Arepresentative magnified (600×) image of border zone myocardiumconfirming the localization of TUNEL signal in the cardiomyocytes.Cardiomyocyte nuclei were counter-stained with DAPI and thecardiomyocyte character was validated by dual staining of the myocardiumwith anti-α sarcomeric actin (FIG. 2C).

The apoptotic signal in border zone myocardium 24 hrs post myocardialinfraction was measured by Caspase 3 activity (FIG. 3A), Caspase 9activity (FIG. 3B), and Caspase 9 protein expression (FIG. 3C). Caspase3 and Caspase 9 activity, as well as Caspase 9 protein expression weresignificantly (p0.0001) reduced in the conditioned media (CM) treatedgroup while the anti-apoptotic protein BCL2 (FIG. 3D) was significantlyupregulated. The anti-apoptotic effects of CM are mediated by TGFβ(p<0.001) and IGF1 (p<0.01), since neutralizing antibodies to TGFβ andIGF1 significantly reduced the anti-apoptotic effects of CM. The dataare expressed as Mean±SEM of border zone myocardium samples from 3-4pigs/group.

The apoptotic signal in border zone myocardium 8 weeks post myocardialinfraction was analyzed. Apoptotic markers, TUNEL positive nuclei (FIG.4A), Caspase 9 activity (FIG. 4B). Caspase 3 activity (FIG. 4C) and BCL2protein expression (FIG. 4D) were not effected at chronic time point (8wks) post conditioned media therapy. The data are expressed as Mean±SEMof border zone myocardium samples from 3-4 pigs/group.

Example 4 Conditioned Media Reduces the Left Ventricular Infarct Area at8 Weeks but not 24 Hrs Post Myocardial Infarction

The effect of conditioned media on left ventricular infarct area wasanalyzed. 24 hours (acute study) or 8 weeks (chronic study) afterinfarct generation, the animals underwent repeat coronary angiography toconfirm infarct related artery TIMI-3 patency, via the left internalcarotid artery, sacrificed and the hearts explanted, weighed andsectioned in 5 mm transverse slices from apex to base (6-8slices/heart). A representative mid ventricular slice (3^(rd) or 4^(th)slice) was stained with 2% TTC.

Conditioned media (CM) therapy appears not to influence the leftventricular infarct area at 24 hrs post MI (FIG. 5A). However, CMtherapy resulted in significant (P<0.01) reduction in the leftventricular infarct area at 8 weeks post MI (FIG. 5B). The data areexpressed as Mean±SEM of 6-7 ventricular cross sections/pig heart with3-4 pigs/group.

Example 5 Conditioned Media Therapy Improves Left Ventricular Function

The function of the left ventricular apex was analyzed 24 hrs and 8weeks post MI. Conditioned media (CM) therapy significantly improved theleft ventricular function i.e., ±dp/dt (FIG. 6A), ±dv/dt (FIG. 6B),stroke volume (FIG. 6C), and Ejection fraction (FIG. 6D) at 24 hrs postMI. The effects of CM appear to be mediated by TGF and IGF1. The dataare expressed as Mean±SEM 20-25 cardiac cycles/pig with 3-4 pigs/group.

Conditioned media (CM) therapy significantly improved the leftventricular function i.e., ±dp/dt (FIG. 7A) ±dv/dt (FIG. 7B), strokevolume (FIG. 7C), and Ejection fraction (FIG. 7D) at 8 weeks post MI.The effects of CM at 8 weeks appear to be less dependent on TGF andIGF1. The data are expressed as Mean±SEM 20-25 cardiac cycles/pig with3-4 pigs/group.

Example 6 Conditioned Media Effects on Border Zone Collagen Content

The effect of Conditioned media (CM) on border zone collagen content at24 hrs and 8 weeks post MI were analyzed.

CM therapy significantly reduced the collagen content in the border zonemyocardium at the acute (24 h, FIG. 8A) but not chronic (8 wks, FIG. 8B)time frame. The border zone sections were stained for collagen usingpicrosirus red and quantified using the NIH imageJ software. Collagenstaining with picrosirus red on 5 sections/pig (n=3-4) with an averageof 5 High power fields/section is expressed as Mean±SEM.

Example 7 Conditioned Media (CM) Increases Border Zone CardiomyocyteHypertrophy at 8 Weeks Post MI

The effects of conditioned media (CM) on borderzone cardiomyocytehypertrophy was analyzed. Cardiomyocytes were incubated withanti-laminin antibody and anti-α sarcomeric actin and subsequentlylabeled. Cell nuclei were stained with DAPI. At least 3 distinctsections from the infarct borderzone in each animal, and a minimum 1000cardiomyocytes were studied in each treatment group.

CM significantly (p<0.001) increased border zone cardiomyocytehypertrophy at 8 weeks post myocardial infarction (as measured by borderzone cell surface area) and this effect was mediated by TGF and IGF1, asneutralizing antibodies to TGF and IGF1 decreased the hypertrphiceffects mediated by CM (FIG. 9). An average of 300-400 border zonecardiomyocytes were analyzed per pig (with 3-4 pigs/group) and the dataare reported as Mean±SEM of 1000-1600 cardiomyocytes/group.

Conditioned media therapy did not affect the total number of nuclei inthe border zone myocardium at 24 h post MI (FIG. 10). Total number ofDAPI positive nuclei/High power field were counted and expressed asMean±SEM of 10 sections/animal.

Example 8

Antiapoptotic effect of conditioned media (CM) therapy on borderzone(BZ) myocardium in vivo (TUNEL staining) was abrogated by blocking IGF-1in CM using selective neutralizing antibody. These data suggest thatIGF-1 is the key cytoprotective factor in CM. The histogram data areexpressed as mean±SEM (n=4). * P<0.01 versus CM treated group. FIG. 11.

Example 9

Effect of conditioned media on left ventricular infarct area at 8 weeks(A) post myocardial infarction (MI) was significantly reduced by CMtherapy, which also increased thinning ratio (B) at 8 weeks post MI.Neutralizing antibody to IGF-1 in CM reversed these so beneficialeffects. Data are expressed as mean±SEM of 7 ventricular crosssections/pig heart (each 5 mm thick) with 4 pigs/group. * P<0.01 versusCM treated group. FIG. 12.

Example 10

Antiapoptotic effect of IGF1 (50 and 500 pg/ml) therapy on borderzone(BZ) myocardium in vivo. Significant reduction in TUNEL staining wasobserved in the BZ myocardium of the IGF1 treated group. All histogramdata are expressed as mean±SEM (n=4-5). * P<0.05 versus control group.FIG. 13.

Example 11 Evaluation of an IGF7 Eluting Stent for its TherapeuticBenefits (Myocardial Infarction Repair) in a Porcine Model of MyocardialInfarction

The experimental protocols are approved by UCC animal ethics committee.Land race pigs (25-30 kgs) are put on aspirin (75 mg/day orally), plavix(75 mg/day orally) and amiodarone (300 mg/day orally) for a week beforesurgery. Anaesthesia is induced by a combination of Xylazine (2 mg/kg),ketamine (15 mg/kg) and glycopyrollate (0.01 mg/kg) and maintained byisofluorane (1.5-2%). The surgical site is cleaned with sterile gauzeand smeared with betadine. A 5 cm incision is made 2 cm lateral andparallel to the trachea and by blunt dissection the carotid artery isexposed. Carotid arterial cut down is performed and interventionalsheath placed to facilitate the insertion of balloon catheter and stentplacement. The Philips fluoroscopy C arm is used to visualize thelocation and guiding the balloon and stent to its appropriate positionin the coronary artery. Myocardial infarction is induced by percutaneousballoon occlusion of the left anterior descending coronary artery for 90minutes followed by 2 hours reperfusion and will be sacrificed at 24hours (acute) or 8 weeks (chronic) respectively. The IGF1 releasingstent is deployed at the site of balloon occlusion with the stent toartery ratio of 1.1:1. After the stent placement the catheters arepulled out and the arteriotomy site and the surgical wound is closed andsmeared with betadine. 24 hr and 8 weeks post procedure the pigs arere-evaluated for myocardial function using pressure-volume loops and CTimaging and then euthanized by over does of pentobarbital. The heart andthe coronary artery with stent are dissected out and processed forhistological examination.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

The invention claimed is:
 1. A method of treating a mammal that hassuffered a myocardial infarction to stimulate repair or survival ofcardiac muscle damaged by the infarct, and to stimulate left ventricularremodeling, the method comprising a step of administering IGF-1 to themammal by intracoronary infusion upstream of the myocardial infarctionin sufficient quantity to reduce the effects of the myocardialinfarction on the mammal by exerting anti-apoptotic and/or cardiotrophiceffects on the affected or diseased tissue, wherein the IGF-1 isadministered in a plurality of intracoronary infusions interspersed withperiods during which the IGF-1 is not infused.
 2. A method as claimed inclaim 1, in which the intracoronary infusion is delivered through anangioplasty balloon.
 3. A method as claimed in claim 1, in which theintracoronary infusion is delivered through an angioplasty balloon, andin which the balloon is inflated for the periods of infusion, anddeflated during the non-infusion periods.
 4. A method as claimed inclaim 1, in which the IGF-1 is delivered at a concentration of from 1 to100 pg/ml.
 5. A method as claimed in claim 1, in which the IFG-1 isadministered over a period of from 5 seconds to one hour.
 6. A method asclaimed in claim 1 in which the IFG-1 is administered over a period offrom 1 to 30 minutes.
 7. A method of treating a mammal that has suffereda myocardial infarction to stimulate repair or survival of cardiacmuscle, and to stimulate left ventricular remodeling, the methodcomprising selecting an individual who has suffered a myocardialinfarction and is in need of left ventricular remodeling, andadministering IGF-1 to the mammal by intracoronary infusion upstream ofthe myocardial infarction in sufficient quantity to reduce the effectsof the myocardial infarction on the mammal by exerting anti-apoptoticand/or cardiotrophic effects on the affected or diseased tissue, whereinthe IGF-1 is administered in a plurality of intracoronary infusionsinterspersed with periods during which the IGF-1 is not infused.